Recombinant vector for expressing target protein in plant cell

ABSTRACT

Provided is a technique for highly expressing a target protein in a plant cell by using a glycosylation domain, a recombinant vector comprising a gene encoding a fusion protein of a glycosylation domain and a target protein, a recombinant cell, a transformed plant, and a method of producing a target protein using these.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a 371 of PCT/KR2018/0000807, filed Jan. 17, 2018,which claims the benefit of priority from Korean Patent Application No.10-2017-0008160, filed Jan. 17, 2017, the contents of each of which areincorporated herein by reference in its entirety.

SEQUENCE LISTING

The Sequence Listing submitted in text format (.txt) filed on Jul. 30,2021, named “SequenceListing.txt”, created on Jul. 30, 2021 (28.2 KB),is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a technique for highly expressing atarget protein in a plant cell by using a glycosylation domain, arecombinant vector comprising a gene encoding a fusion protein of aglycosylation domain and a target protein, a recombinant cell, atransgenic plant, and a method of producing a target protein usingthese.

BACKGROUND ART

The remarkable development of molecular biology and genetic engineeringtechniques has also been applied to the plant field, and efforts toproduce useful physiologically active substances from plants aresteadily continuing. When producing useful substances in plants,production costs may be dramatically reduced, various contaminants suchas viruses, oncogenes, and enterotoxins that may be generated in aconventional method of separating and purifying a protein throughsynthesis in animal cells or microorganisms may be fundamentallyexcluded, and unlike animal cells or microorganisms, such usefulsubstances may be stored and managed as seeds for a long period of timeeven in the commercialization stage. In addition, when demand for thecorresponding useful substance surges, the above system is absolutelyadvantageous compared to existing animal cell systems in terms ofequipment technology or costs required for mass production, and thussupply corresponding to the increased demand is possible within theshortest time.

Despite these advantages, however, a relatively low level of proteinexpression is the biggest drawback in protein production in plant cells,compared to other hosts including animal cells. Thus, many studies havebeen conducted and there have been attempts to increase a proteinexpression level in plant cells by using various methods.

Previous studies to increase a level of target protein expression in aplant cell have been focused mainly on a transcription stage prior to atranslation stage where a protein is produced from mRNA during a proteinexpression process, and few studies have been conducted on a method ofincreasing a protein expression level when a protein is translated frommRNA.

Meanwhile, it is well known that conventional N-glycosylation affectsprotein stability. In this case, N-glycans were thought to enhancestability because they protect proteins from proteases. It is also knownthat other mechanisms provide an additional binding force for proteinthree-dimensional structures to thereby provide stability.

As a result of having made intensive efforts to increase an expressionlevel of a target protein in the translation stage in producing a targetprotein in a plant cell, the inventors of the present inventionconfirmed that, when a small domain causing glycosylation is fused to atarget protein, an expression level of the protein was increased, andverified that production efficiency of the target protein could beincreased in a transgenic plant by using the above finding, thuscompleting the present invention.

DISCLOSURE Technical Problem

The present invention relates to a use of an N-glycosylation domain intarget protein expression.

An embodiment provides a composition for expressing a target protein,which comprises one or more selected from the group consisting of a geneencoding an N-glycosylation domain, a recombinant vector comprising thegene, and a recombinant cell comprising the recombinant vector.

The composition for expressing a target protein may further comprise agene encoding the target protein or a recombinant vector comprising thegene. In this regard, the gene encoding the target protein and the geneencoding an N-glycosylation domain may be comprised in the form of agene encoding a fusion protein comprising the N-glycosylation domain andthe target protein or a recombinant vector comprising the gene.

The N-glycosylation domain may be an N-glycosylation domain (e.g.,multiple N-glycosylation domains) comprising one or more N-glycosylationsites or two or more N-glycosylation sites. In one embodiment, theN-glycosylation domain may comprise a CD45-derived M domain, such as ahuman CD45-derived M domain or a portion thereof. The target proteinexpression may be performed in a eukaryotic cell (e.g., a plant cell) ora eukaryotic organism (e.g., a plant).

Another embodiment provides a recombinant vector comprising a geneencoding a fusion protein comprising an N-glycosylation domain and atarget protein.

Another embodiment provides a recombinant cell into which therecombinant vector is introduced. The recombinant cell may be aeukaryotic cell, for example, a plant cell.

The recombinant vector comprising a gene encoding a fusion proteinand/or the recombinant cell may be used for enhancing production of thetarget protein. Therefore, another embodiment provides a composition forproducing a target protein or enhancing production of a target protein,which comprises a recombinant vector comprising a gene encoding a fusionprotein comprising an N-glycosylation domain and a target protein and/ora recombinant cell.

Another embodiment provides a transgenic organism into which therecombinant vector comprising a gene encoding a fusion proteincomprising an N-glycosylation domain and a target protein is introduced.The transgenic organism may be a transgenic eukaryotic organism, forexample, a transgenic plant.

Another embodiment provides a method of producing a target protein orenhancing production of a target protein, comprising introducing, into acell, the composition for producing a target protein or enhancingproduction of a target protein. The method may increase an expressionlevel or productivity of the target protein, compared to a case in whicha gene encoding the target protein is introduced alone into a cell(i.e., introduced via an N-glycosylation domain-free recombinantvector).

Technical Solution

The present invention has been made to address the above-describedproblems, and provides a use of an N-glycosylation domain for expressinga target protein in a plant, more particularly, a technique forincreasing an expression level of a target protein by expressing afusion gene produced by fusing a gene encoding the target protein and agene encoding an N-glycosylation domain to a C-terminal-correspondingsite (3′-terminal) or N-terminal-corresponding site (5′-terminal) of thegene.

An embodiment provides a composition for expressing a target protein,which comprises one or more selected from the group consisting of a geneencoding an N-glycosylation domain, a recombinant vector comprising thegene, and a recombinant cell comprising the recombinant vector.

The composition for expressing a target protein may further comprise agene encoding the target protein or a recombinant vector comprising thegene. In this regard, the gene encoding the target protein and the geneencoding an N-glycosylation domain may be comprised in the form of agene encoding a fusion protein comprising the N-glycosylation domain andthe target protein or a recombinant vector comprising the gene.

The N-glycosylation domain may be an N-glycosylation domain comprisingone or more N-glycosylation sites or two or more N-glycosylation sites.In one embodiment, the N-glycosylation domain may comprise aCD45-derived M domain or a portion thereof, for example, a humanCD45-derived M domain or a portion thereof. The target proteinexpression may be performed in a eukaryotic cell (e.g., a plant cell) ora eukaryotic organism (e.g., a plant).

Another embodiment provides a recombinant vector comprising a geneencoding a fusion protein comprising an N-glycosylation domain and atarget protein. The recombinant vector may be used for expression in aeukaryotic cell, for example, a plant cell.

Another embodiment provides a recombinant cell comprising the geneencoding a fusion protein comprising an N-glycosylation domain and atarget protein. The recombinant cell may be a cell into which arecombinant vector comprising a gene encoding a fusion proteincomprising an N-glycosylation domain and a target protein is introduced.The cell may be a eukaryotic cell, for example, a plant cell.

Another embodiment provides a transgenic organism comprising the geneencoding a fusion protein comprising an N-glycosylation domain and atarget protein. The transgenic organism may be an organism into which arecombinant vector comprising a gene encoding a fusion proteincomprising an N-glycosylation domain and a target protein is introduced.The transgenic organism may be a transgenic eukaryotic organism, forexample, a transgenic plant. The transgenic organism may be a eukaryoticorganism (e.g., a plant) comprising the above-described recombinantcell.

The recombinant vector comprising a gene encoding a fusion proteinand/or the recombinant cell and/or the transgenic organism may be usedfor producing a target protein or enhancing the production of a targetprotein.

Therefore, another embodiment provides a composition for producing atarget protein or enhancing the production of a target protein, thecomposition comprising one or more selected from the group consisting ofa gene encoding a fusion protein comprising an N-glycosylation domainand a target protein, a recombinant vector comprising the gene, arecombinant cell comprising the recombinant vector, and a transgenicorganism comprising the recombinant vector.

Another embodiment provides a method of producing a target protein orenhancing the production of a target protein, comprising culturing arecombinant cell comprising a gene encoding a fusion protein comprisingan N-glycosylation domain and a target protein. The recombinant cell maybe a cell into which a recombinant vector comprising a gene encoding afusion protein comprising an N-glycosylation domain and a target proteinis introduced. The cell may be a eukaryotic cell, for example, a plantcell. The method may further comprise, before the culturing process,introducing, into a cell, a recombinant vector comprising a geneencoding a fusion protein comprising an N-glycosylation domain and atarget protein. The method may further comprise, after the culturingprocess, isolating (or extracting) and/or purifying a target proteinfrom the cultured cell (a cell, cell debris, or a cell lysate) and/or aculture medium.

Another embodiment provides a method of producing a target protein orenhancing the production of a target protein, comprising growing atransgenic organism comprising a gene encoding a fusion proteincomprising an N-glycosylation domain and a target protein. The organismmay be a eukaryotic organism, for example, a plant. The method mayfurther comprise, before the growing process, introducing, into anorganism, a recombinant vector comprising a gene encoding a fusionprotein comprising an N-glycosylation domain and a target protein. Themethod may further comprise, after the growing process, isolating (orextracting) and/or purifying a target protein from the eukaryoticorganism (e.g., a plant), or a cell of the eukaryotic organism (a cell,cell debris, cell lysate or a culture of the cell).

The method may increase an expression level or productivity of thetarget protein, compared to a case in which a gene encoding the targetprotein is introduced alone into a cell or an organism (i.e., introducedvia an N-glycosylation domain-free recombinant vector), a case in whicha target protein fused with an O-glycosylation domain is used, and/or acase in which the target protein intrinsically contains anN-glycosylation site without being fused with a separate N-glycosylationdomain.

In one embodiment, the N-glycosylation domain may be a polypeptidehaving one or more N-glycosylation sites or two or more N-glycosylationsites (N-glycosylated amino acid; asparagine (Asn)), for example, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 N-glycosylation sites and having a totalnumber of amino acids of 10 to 100 or 20 to 80. In one embodiment, theN-glycosylation domain may be a polypeptide comprising 10 to 100 or 20to 80 consecutive amino acids comprising at least a human CD45-derived Mdomain (having 4 N-glycosylation sites) in human CD45 or a portionthereof. For example, the human CD45 protein may have an amino acidsequence represented by SEQ ID NO: 5 (UniProt No. P08575). The humanCD45-derived M domain may be a polypeptide consisting of a total of 60amino acids from Ala (residue 231) to Asp (residue 290) of the humanCD45 protein (SEQ ID NO: 5) (60aa; ANITVDYLYN KETKLFTAKL NVNENVECGNNTCTNNEVHN LTECKNASVS ISHNSCTAPD; SEQ ID NO: 2; underlined and boldcharacters denote N-glycosylation sites). In one embodiment, a geneencoding the human CD45-derived M domain may comprise a nucleic acidsequence of SEQ ID NO: 1. A portion of the human CD45-derived M domainmay be a fragment of a polypeptide comprising 10 or more, 15 or more, or20 or more consecutive amino acids having one or more N-glycosylationsites or two or more N-glycosylation sites, for example, 1, 2, 3, or 4N-glycosylation sites in the CD45-derived M domain (e.g., selected fromN(Asn) at residue 2, N(Asn) at residue 30, N(Asn) at residue 40, andN(Asn) at residue 46 of SEQ ID NO: 2). The fragment of the polypeptidemay be a polypeptide comprising 10 or more, 15 or more, or 20 or moreconsecutive amino acids having one or more amino acid residue selectedfrom N at residue 40 and N at residue 46 of the amino acid sequence ofSEQ ID NO: 2, and may be, for example, “LTECKNASVS ISHNSCTAPD (SEQ IDNO: 6)” or “NVNENVECGN NTCTNNEVHN LTECKNASVS ISHNSCTAPD (SEQ ID NO: 7)”,but the present invention is not limited thereto. In one embodiment, theN-glycosylation domain may be a polypeptide (e.g., SEQ ID NO: 6, 7, or8) comprising 10 to 100 or 20 to 80 consecutive amino acids having an Mdomain (SEQ ID NO: 2) or an M domain portion comprising 10 or more, 15or more, or 20 consecutive amino acids of the M domain (SEQ ID NO: 2),in the human CD45 protein (SEQ ID NO: 5). In one embodiment, a geneencoding the M domain may have a nucleic acid sequence represented bySEQ ID NO: 1.

In one embodiment, the recombinant vector may further comprise one ormore selected from the group consisting of a transcriptional regulatoryfactor, a translational regulatory factor, and a marker for confirminggene expression.

In one embodiment, the transcriptional regulatory factor may be one ormore selected from all transcription factors commonly used fortranscriptional regulation in a cell, for example, a plant cell and maybe, for example, one or more selected from the group consisting of acauliflower mosaic virus 35S RNA promoter, a cauliflower mosaic virus19S RNA promoter, a figwort mosaic virus-derived full-lengthtranscription promoter, and a tobacco mosaic virus coat proteinpromoter, but the present invention is not limited thereto.

The translational regulatory factor may be one or more selected from alltranslational regulatory factors commonly used for translationalregulation in a cell, for example, a plant cell and may be, for example,an M17 factor, but the present invention is not limited thereto. The M17factor may have a nucleic acid sequence represented by SEQ ID NO: 3.

In another embodiment, the recombinant vector may further comprise asignal sequence for targeting (migration and/or retention) to a specificintracellular organelle. In one embodiment, the recombinant vector maybe engineered to target the endoplasmic reticulum (ER), and to this end,may further comprise an endoplasmic reticulum (e.g., a cell membranesurface) transfer signal (e.g., a BiP (chaperone bindingprotein)-encoding gene or the like) and/or an endoplasmic reticulumretention signal (e.g., HDEL (His-Asp-Glu-Leu) (SEQ ID NO: 54)peptide-encoding gene). The signal sequence for targeting anintracellular organelle (e.g., an endoplasmic reticulum) may be linkedto the N-terminal (5′-terminal of a gene encoding a fusion protein) orC-terminal (3′-terminal of the gene encoding a fusion protein), forexample, N-terminal (5′-terminal of a gene encoding a fusion protein) ofthe fusion protein. As such, the recombinant vector may be targeted tothe endoplasmic reticulum (e.g., inside the endoplasmic reticulum) ofthe intracellular organelle, thereby further increasing a proteinexpression level (see FIG. 3). In one embodiment, N-glycosylation of thefusion protein may occur in the endoplasmic reticulum.

The BiP (chaperone binding protein) may have a nucleic acid sequencerepresented by SEQ ID NO: 4.

In one embodiment, the present invention provides a recombinant vectorfor transforming a plant to increase an expression level of a targetprotein, the recombinant vector comprising a gene encoding the targetprotein, a gene encoding a human CD45-derived M domain, atranscriptional regulatory factor, an M17 factor operably linked to thetranscriptional regulatory factor, and a gene encoding BiP (chaperonebinding protein) and/or a HDEL (His-Asp-Glu-Leu) peptide.

In one embodiment, the recombinant vector may further comprise a markerfor confirming gene expression. In one embodiment of the presentinvention, a HA epitope sequence was used to confirm the presence orabsence of expression by western blotting, but the present invention isnot limited thereto.

The eukaryotic cell described herein may be one or more selected fromthe group consisting of a fungus, an animal cell, and a plant cell, andmay be, for example, a plant cell. The eukaryotic organism may be one ormore selected from the group consisting of all unicellular eukaryoticorganisms and multicellular eukaryotic organisms (plants or animals) andmay be, for example, a plant. The plant described herein may be one ormore plant selected from all algae, monocotyledonous plants, anddicotyledonous plants, or a cell thereof and may be, for example, adicotyledonous plant selected from the group consisting of Arabidopsis,soybeans, tobacco, eggplants, peppers, potatoes, tomatoes, Koreancabbage, radish, cabbage, lettuce, peaches, pears, strawberries,watermelons, melons, cucumbers, carrots, and celery; a monocotyledonousplant selected from rice, barley, wheat, rye, corn, sugarcane, oats, andonions; or a cell thereof, but the present invention is not limitedthereto.

The introduction of the recombinant vector into a eukaryotic organism(e.g., a plant) or a eukaryotic cell (e.g., a plant cell) may beperformed using a general transduction method, for example, using one ormore methods selected from the group consisting of an Agrobacteriumsp.-mediated method, particle gun bombardment, silicon carbide whiskers,sonication, electroporation, and polyethylene glycol (PEG)-mediatedtransformation, but the present invention is not limited thereto.

As described above, the term “N-glycosylation” as used herein refers toa series of processes for binding glycans, which are sugar moleculeoligosaccharides, to the nitrogen atom of an amino acid of a protein,and is distinguished from 0-glycosylation, which binds sugar moleculesto the oxygen atom of an amino acid residue of a protein. In the presentspecification, N-glycosylation may occur in an endoplasmic reticulum(e.g., inside an endoplasmic reticulum).

The term “N-glycosylation domain” as used herein refers to a polypeptidecomprising an N-glycosylation site (amino acid residue), which may benon-naturally occurring, e.g., chemically or recombinantly synthesized,or naturally occurring.

In one embodiment, a recombinant vector comprising a gene encoding afusion protein produced by fusing a human CD45-derived M domain to theC-terminal of a target protein was prepared to be used in experiments,but the present invention is not limited thereto, and the humanCD45-derived M domain may also be fused to the N-terminal of a targetprotein.

The fusion protein comprising a target protein and an N-glycosylationdomain may comprise a suitable linker (e.g., a 1-50 aa, 1-30 aa, 1-20aa, 2-50 aa, 2-30 aa, or 2-20 aa peptide linker) between the targetprotein and the N-glycosylation domain. The peptide linker may be asequence in which glycine-serine is repeated, but the present inventionis not limited thereto.

The amino acid sequences and nucleic acid sequences described herein maybe construed as extending to a sequence with at least 70% homology, atleast 75% homology, at least 80% homology, at least 85% homology, atleast 90% homology, at least 91% homology, at least 92% homology, atleast 93% homology, at least 94% homology, at least 95% homology, atleast 96% homology, at least 97% homology, at least 98% homology, or atleast 99% homology, to the provided sequences.

The “% sequence homology” may be confirmed by comparing two optimizedsequences using a comparison region, and some of the polynucleotidesequences in the comparison region may comprise an addition or deletion(i.e., a gap) compared to reference sequences (additions or deletionsexcluded) for the optimal alignment of two sequences.

The term “recombinant vector or recombinant cell” as used herein refersto a cell that replicates a heterologous nucleic acid (polynucleotide)or expresses the nucleic acid, or a vector or cell that expresses aprotein encoded by a peptide, a heterologous peptide, or a heterologousnucleic acid. The recombinant cell may express a gene or gene segmentthat is not found in a natural form of the cell in one of a sense formand/or an antisense form. In addition, the recombinant cell may expressa gene found in a cell in its natural state, but the gene is a modifiedform and a gene reintroduced into a cell by an artificial means.

The “recombinant vector” may be one or more selected from the groupconsisting of all plasmids, phage, yeast plasmids, plant cell viruses,mammalian cell viruses, and other media known in the art into which agene sequence or nucleotide sequence can be inserted or introduced.Generally, any plasmid and vector may be used without particularlimitation as long as it is capable of replicating and being stabilizedin a plant cell or a plant host. In one embodiment, the recombinantvector may be for use in transforming a plant or a plant cell. The genesequence or nucleotide sequence according to the present invention maybe operably linked to an expression regulatory factor, and theexpression regulatory factor operably linked to the gene sequence may becomprised in a single expression vector comprising both a selectablemarker and a replication origin.

Examples of known vectors comprise pBI121, pHellsgate8, pROKII, pBI76,pET21, pSK(+), pLSAGPT, pUC, and pGEM. In addition, examples of vectorsexpressed in plants, which comprises a CMV35s promoter, comprise thepCAMBIA series (pCAMBIA1200, 1201, 1281, 1291, 1300, 1301, 1302, 1303,1304, 1380, 1381, 2200, 2201, 2300, 2301, 3200, 3201, and 3300), pMDC32,and pC-TAPapYL436, but the present invention is not limited thereto.

The term “operably linked” as used herein may refer to a gene and anexpression regulatory factor that are linked in such a way to enablegene expression when an appropriate molecule is bound to the expressionregulatory factor.

The term “expression regulatory factor” as used herein refers to a DNAsequence that regulates the expression of a polynucleotide sequenceoperably linked in a particular host cell. Such regulatory factorscomprise a transcriptional regulatory factor comprising a promoter forperforming transcription and any operator sequence, a translationalregulatory factor comprising a sequence encoding an appropriate mRNAribosome-binding site and a sequence that regulates protein synthesis,and a sequence that regulates the termination of transcription andtranslation.

In the present invention, the transcriptional regulatory factor may beselected from the group consisting of a cauliflower mosaic virus 35S RNApromoter, a cauliflower mosaic virus 19S RNA promoter, a figwort mosaicvirus-derived full-length transcription promoter, and a tobacco mosaicvirus coat protein promoter, but the present invention is not limitedthereto.

In the present invention, the translational regulatory factor may be anM17 sequence, which serves to increase the amount of a target proteinsynthesized in a plant. In the present invention, preferably, the M17sequence may be represented by SEQ ID NO: 3.

The recombinant vector of the present invention may further comprise anucleic acid encoding BiP (chaperone binding protein) or a HDEL(His-Asp-Glu-Leu) peptide, which may be operably linked to thetranscriptional regulatory factor.

The BiP, which is a luminal binding protein, was identified as animmunoglobulin heavy chain binding protein and a glucose regulatedprotein, and is a member of the HSP70 chaperone family located in theendoplasmic reticulum and temporarily binds to a protein newlysynthesized in the endoplasmic reticulum. In addition, BiP serves toenable target proteins to be targeted to the endoplasmic reticulum sinceit has, at the N-terminal thereof, a signal sequence that determinestargeting to the endoplasmic reticulum. For example, a nucleic acidencoding the BiP may have a nucleic acid sequence represented by SEQ IDNO: 4.

In addition, the recombinant vector for transforming a plant maycomprise a nucleotide sequence encoding an ER retention signal peptidesuch as HDEL. In the case of a HDEL signal peptide, a target protein isretained in the ER such that folding and assembly by a molecularchaperone are increased, resulting in further minimization of proteindegradation. As an example, it is known that, in a case in which atarget protein is retained more in the ER when sent to the secretorypathway, a yield of the target protein is increased about 10-fold toabout 100-fold.

An embodiment also provides a recombinant vector for transforming aplant to increase an expression level of a target protein, therecombinant vector comprising a target protein, a gene encoding a humanCD45-derived M domain or a portion thereof, a transcriptional regulatoryfactor, M17 operably linked to the transcriptional regulatory factor,and a nucleic acid encoding BiP (chaperone binding protein) and a HDEL(His-Asp-Glu-Leu) (SEQ ID NO: 54) peptide.

The recombinant vector described herein may further comprise a markerfor confirming gene expression. In one embodiment, a HA epitope sequencewas used to confirm expression by western blotting, but the presentinvention is not limited thereto.

The term “target protein” as used herein refers to a protein forproducing or a fragment thereof, and the target protein is not limitedto a specific protein. Specifically, the target protein may be any oneor more selected from the group consisting of an antigen, an antibody,an antibody fragment, a structural protein, a regulatory protein, atranscription factor, a toxin protein, a hormone, a hormone analog, acytokine, an enzyme, an enzyme inhibitor, a transport protein, areceptor (e.g., tyrosine kinase receptor and the like), a receptorfragment, a biological defense inducer, a storage protein, a movementprotein, an exploitive protein, a reporter protein, and the like.

In one embodiment, leptin, GLP-1, Exendin-4, aprotinin, a greenfluorescent protein (GFP), and the like have been described as examplesof the target protein, but these are provided for illustrative purposesonly to achieve a protein production-enhancing effect provided in thepresent specification, but the target protein is not limited to theabove-listed proteins.

The present invention also provides a plant transformed with therecombinant vector. The transformed plant comprises an M domain sequenceand is devised to be operably linked to transcriptional andtranslational regulatory factors and controlled thereby. The transformedplant described herein may be a whole plant, a plant cell (e.g., a cellsuch as a leaf, a stem, a root, and the like), or plant tissue (e.g., aleaf, a stem, a root, and the like). The plant tissue may comprise aplant seed. The plant may be an herbaceous or textured plant, and may bea dicotyledonous plant or a monocotyledonous plant. In particular, thedicotyledonous plant may be selected from the group consisting ofArabidopsis, soybeans, tobacco, eggplants, peppers, potatoes, tomatoes,Korean cabbage, radish, cabbage, lettuce, peaches, pears, strawberries,watermelons, melons, cucumbers, carrots, and celery, and themonocotyledonous plant may be selected from the group consisting ofrice, barley, wheat, rye, corn, sugarcane, oats, and onions, but thepresent invention is not limited thereto.

A method of introducing the recombinant vector of the present inventioninto a plant may be selected from an Agrobacterium sp.-mediated method,particle gun bombardment, silicon carbide whiskers, sonication,electroporation, and polyethylene glycol (PEG)-mediated transformation,but the present invention is not limited thereto.

The transformed plant may be obtained through a sexual propagationmethod or an asexual propagation method, which is a conventional methodin the art. More specifically, the plant of the present invention may beobtained through sexual propagation, which is a process of producingseeds through pollination and propagating from the seeds. In addition,the plant may be transformed with the recombinant vector according tothe present invention and then obtained through asexual propagation,which is a process of inducing callus, rooting, and acclimatizing soil,according to a conventional method. That is, an explant of the planttransformed with the recombinant vector according to the presentinvention is placed in a suitable medium known in the art, and thencultured under appropriate conditions to induce callus formation, andwhen shoots are formed, they are transferred to a hormone-free mediumand cultured. After about 2 weeks, the shoots are transferred to arooting medium to induce roots. Thereafter, the roots may betransplanted into the soil and acclimatized, thereby obtaining the plantaccording to the present invention. The transformed plant of the presentinvention may comprise tissues, cells, or seeds obtainable therefrom.

The present invention also provides a method of producing a targetprotein, comprising: constructing a recombinant vector for transformingthe plant; introducing the recombinant vector into a plant to produce atransgenic plant; culturing the transgenic plant; and isolating andpurifying a target protein from the transgenic plant or a culturesolution.

The introduction of the recombinant vector into a plant cell or a plantmay be performed using one or more methods selected from the groupconsisting of an Agrobacterium sp.-mediated method, particle gunbombardment, silicon carbide whiskers, sonication, electroporation, andpolyethylene glycol (PEG)-mediated transformation. In one embodiment ofthe present invention, PEG-mediated transformation was used.

Advantageous Effects

A recombinant vector for transforming a plant, according to the presentinvention, has overcome the difficulty of obtaining a highly expressedtransformant, which was the biggest problem in protein production usingexisting plant transformation, by increasing an expression level of atarget protein in a plant cell, and thus is expected to be great help inthe production of useful proteins using a plant.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram illustrating a linkage comprising a p35S-M17: Bip:Leptin: M: HA: HDEL moiety in a recombinant vector for transforming aplant which was constructed according to an embodiment.

FIG. 2A is a diagram illustrating a linkage comprising a p35S-M17: Bip:Leptin: HA: HDEL moiety, a p35S-M17: Bip: Leptin: M: HA: HDEL moiety,and a p35S-M17: Bip: Leptin: M1234: HA: HDEL moiety in a recombinantvector for transforming a plant which was constructed according to anembodiment (M1234: M domain variant in which N-glycosylation sites N1,N2, N3, and N4 of an M domain were modified (Asn→Gln)).

FIG. 2B illustrates electrophoresis results (upper side) of confirmingprotein expression levels by western blotting according to the presenceor absence of N-glycosylation of an M domain and quantification resultsthereof (lower side).

FIG. 3 is a diagram of a recombinant vector for testing a differencebetween protein expression levels according to a targeted plant cellorganelle (upper side), and illustrates electrophoresis results (middleside) of confirming protein expression levels by western blotting andquantification results thereof (lower side).

FIG. 4 illustrates electrophoresis results (upper side) of confirmingexpression patterns of fusion proteins fused with an M domain over timeby western blotting and quantification results thereof (lower side).

FIG. 5 illustrates western blotting results of confirming an expressionlevel of a fusion protein produced by fusing an M domain with Exendin4or GLP-1.

FIG. 6A is a diagram of a recombinant vector for expressing fusionproteins produced by fusing an M domain and various target proteins invarious orders, according to an embodiment.

FIG. 6B illustrates western blotting results of confirming an expressionlevel of a fusion protein produced by fusing an M domain and leptin(Lep), aprotinin (Apr), or GFP (Gfp) in various orders.

FIGS. 7A-7C is a set of graphs showing expression levels of fusionproteins in which Leptin was fused with each of mutant M domains wherethe N-glycosylation site of an M domain was mutated in variouscombinations.

FIG. 8 illustrates western blotting results of confirming an expressionlevel of a fusion protein fused with a wild type M domain or a mutant Mdomain according to the presence or absence of ER-associated degradationinhibition.

FIG. 9 illustrates western blotting results of confirming proteinexpression levels according to the presence or absence of fusion of atarget protein intrinsically having an N-glycosylation site with an Mdomain.

FIG. 10 illustrates western blotting results of confirming expressionlevels of fusion proteins produced by fusing a target protein withvarious M domain portions or extension portions.

FIG. 11 is a graph showing quantitative RT-PCR results of confirmingtranscription levels according to fusion of an M domain or various Mdomain variants.

MODE OF THE INVENTION

Hereinafter, the present invention will be described in further detailwith reference to the following examples.

It will be obvious to those of ordinary skill in the art that theseexamples are provided for illustrative purposes only and are notintended to limit the scope of the present invention.

Reference Example 1: Preparation of Plant Materials

Arabidopsis (Arabidopsis thaliana ecotype, Col-0) plants were grown onB5 plates in a growth chamber at 20° C. to 22° C. under a 16 h/8 hlight/dark cycle. Leaf tissues from 2-week-old plants were used forprotoplast isolation.

Reference Example 2: Plasmid Construction

The mature peptide region of mouse leptin cDNA (NM_008493.3) was used. ADNA fragment (SEQ ID NO: 1) encoding an M domain was synthesized byrepetitive PCR, and mutants of the N-glycosylation (Asn-Glnsubstitution) were generated by PCR-based site-directed mutagenesis. ADNA fragment (SEQ ID NO: 11) encoding aprotinin was produced by chemicalsynthesis (Bioneer, Daejeon, Korea). Enterokinase and furin cleavagesites were included in the primer used for leptin amplification(5′-GGATCCAAGATGATGATGATAAGGTGCCTATCCAGAAAGTCCAGGAT-3′ (SEQ ID NO: 18)).AHA epitope and an ER retention signal HDEL were introduced using theprimers used for amplification of the M domain. PCR conditions were asfollows: 94° C. for 5 minutes, (94° C. for 30 seconds, 52° C. for 1minute, and 72° C. for 30 seconds) repeated 30 times, 72° C. for 7minutes. The primer sequences used are summarized in Table 1 below:

TABLE 1  SEQ ID Primer Name Sequence (5′ to 3′) NO. BamHI-Ek-GGATCCAAGATGATGATGATAAGGTGCCTA 18 leptin-F TCCAGAAAGTCCAGGATleptin-furin-  ACTAGTTCGCCTGACACGGCATTCAGGGCT 19 SpeI-R AACATCCAACTGhspt-R GAATTCCTTATCTTTAATCATATT 20 M-3-HA-HDEL-FTCATAATTCATGTACTGCTCCTGATTACCCA 21 TACGATGTTCCAGATTACGCTTCCCACGATGAGCTCTAGCTCGAGATATGAAGATGAAGAT GAAATATT M-2-FAATGTGGAAACAATACTTGCACAAACAATG 22 AGGTGCATAACCTTACAGAATGTAAAAATGCGTCTGTTTCCATATCTCATAATTCATGTAC TGCTCCTGA SpeI-M-1-FACTAGTGCAAACATCACTGTGGATTACTTA 23 TATAACAAGGAAACTAAATTATTTACAGCAAAGCTAAATGTTAATGAGAATGTGGAATGT GGAAACAATACTTGCACAA SpeI-HA-FACTAGTTACCCATACGATGTTCCAGATTAC 24 XbaI-Cab-FTCTAGAATGGCGTCGAACTCGCTTATGAGC 25 Cab-BamHI-R GGATCCTCTCTGACTCTTTGTA 26XbaI-F1-F TCTAGAATGGCAATGGCTGTTTTCCGTCGC 27 F1-BamHI-RGGATCCTCTGAACTGCTCTAAGCTTGGAAG 28 SpeI-M-F ACTAGTGCAAACATCACTGTGGAT 29SpeI-M-N2Q-F ACTAGTGCACAAATCACTGTGGAT 30 M-N30Q-FGTGGAATGTGGACAAAATACTTGCACA 31 M-N30Q-R TGTGCAAGTATTTTGTCCACATTCCAC 32M-N40Q-F AATGAGGTGCATCAACTTACAGAATGT 33 M-N40Q-RACATTCTGTAAGTTGATGCACCTCATT 34 M-N46Q-F ACAGAATGTAAACAAGCGTCTGTTTCC 35M-N46Q-R GGAAACAGACGCTTGTTTACATTCTGT 36 M-N40, 46Q-FAACAATGAGGTGCATCAACTTACAGAATGT 37 AAACAAGCGTCTGTTTCCATA M-N40, 46Q-RTATGGAAACAGACGCTTGTTTACATTCTGTA 38 AGTTGATGCACCTCATTGTT BamHI-M-FGGATCCCGATGGCAAACATCACTGTGGATT 39 ACTTA M-GS2-SpeI-RACTAGTTGATCCACCACCAGACCCACCTCC 40 ACCATCAGGAGCAGTACATGAATTATBamHI-Ek-Apr GGATCCCGGATGACGACGATAAGCGACCGG 41 AC BamHI-ek-Apr-FGGATCCAAGATGATGATGATAAGCGACCGG 42 AC Apr-fu-SpeI-RACTAGTTCGCCTGACACGGGCACCGCCGCA 43 GGTTCTCATACA GFP-HDEL-stop-CTCGAGCTAGAGCTCATCGTGCTTGTACAG 44 XhoI-R CTCGTCCATGCCGAG GFP-fu-SpeI-RACTAGTTCGCCTGACACGCTTGTACAGCTC 45 GTCCATGCCGAG SpeI-ek-GFP-FACTAGTGATGACGACGATAGGTGAGCAAG 46 AtACT2-5′ TATGAATTACCCGATGGGCAAG 47AtACT2-3′ TGGAACAAGACTTCTGGGCAT 48 leptin-F-TCGGTATCCGCCAAGCAGTGCCTATCCAGA 49 qRT1-F AAGTCCA leptin-R-GGTGAAGCCCAGGAATGAAGGCATTCAGG 50 qRT1-R GCTAACATCCA

The mature region of leptin and the M domain or Asn-to-Gln-substitutedmutant M domain were sequentially ligated into the vector BiP: HA: CBD:HDEL.

To accumulate fusion proteins in chloroplasts and mitochondria, a Cabtransit peptide or F1-ATPase gamma subunit presequence was amplified byPCR and substituted with BiP in EeLepf and EeLepfM vectors (see Lee, D.,et al. W. et al., Arabidopsis nuclear-encoded plastid transit peptidescontain multiple sequence subgroups with distinctivechloroplast-targeting sequence motifs. Plant Cell 20, 1603-1622 (2008);Lee, S. et al., Mitochondrial targeting of the Arabidopsis F1-ATPasegamma-subunit via multiple compensatory and synergistic presequencemotifs. Plant Cell 24, 5037-5057 (2012)).

The constructs were all constructed from the same vector and thereforehave the same 5′-UTRs. Nucleotide sequences of all PCR products wereconfirmed by nucleotide sequencing.

Reference Example 3: Expression, Compound Treatment, and WesternBlotting Analysis

The plasmid prepared in Reference Example 2 was introduced into aprotoplast of plant cells prepared in Reference Example 1 bypolyethylene glycol (PEG)-mediated transformation. After transformation,proteins were extracted at 24 hours or at a predetermined time toprepare protein extracts. Immediately after transformation, protoplastswere treated with tunicamycin (10 μg/mL; Sigma-Aldrich, St. Louis, Mo.)and then treated with cycloheximide (50 μg/mL; Sigma-Aldrich, St. Louis,Mo.) 12 hours after transformation. Western blotting analysis wasperformed on protein extracts using an anti-HA antibody (RocheDiagnostics, Indianapolis, Ind.), an anti-actin antibody (MPBiomedicals, Solon, Ohio), an anti-GFP antibody (Bio-Application,Pohang, Korea), or an anti-BiP antibody. Protein blots were developedwith an ECL kit (Amersham Pharmacia Biotech, Piscataway, N.J.) andimages were acquired using a LAS4000 image analyzer (Fujifilm, Tokyo,Japan).

Reference Example 4: Total RNA Isolation and Quantitative RT-PCRAnalysis at Transcript Level

Total RNA was extracted from PEG-mediated transformed plant protoplastsusing an Ambion phenol-free total RNA isolation kit and treated withTURBO DNase (Ambion). cDNA was synthesized from the extracted total RNAusing a high-capacity cDNA reverse transcription kit (AppliedBiosystems). Transcript levels were detected using the Power SYBR GreenPCR Master Mix (Applied Biosystems). ACTIN2 was used as an endogenouscontrol. A PCR mixture (20 μl) contained 50 ng of a template, 0.5 mMforward and reverse primers, and 1×SYBR Mix.

PCR conditions were as follows: initial denaturation at 95° C. for 10min, followed by 40 cycles of 95° C. for 15 s and 60° C. for 1 min.

To confirm specific amplification, a melting curve was generated byheating at 95° C. for 15 s and then at 60° C. for 1 min, and thenincreasing the temperature 0.3° C. every 15 sup to 95° C.

Example 1. Construction of Recombinant Vector for M Domain Fusion

To highly express a target protein in a plant, a recombinant vector forplant transformation which comprised a gene encoding a fusion protein inwhich the M domain of human CD45 was fused to the target protein wasconstructed. As the target protein, Leptin in which an enterokinasecleavage site and a furin cleavage site were fused to the N-terminal andthe C-terminal, respectively (hereinafter referred to as “eLepf”) wasused. A CaMV 35S promoter was used in the vector pCAMBIA1300 as therecombinant vector, which is a commonly used vector, an M17 sequence(SEQ ID NO: 3) was added to increase the amount of protein synthesized,the target protein was transferred to an endoplasmic reticulum using agenomic DNA sequence (SEQ ID NO: 4) corresponding to a signal peptide ofBiP (chaperone binding protein), and HDEL (His-Asp-Glu-Leu) (SEQ ID NO:54) was added to the C-terminal to be retained in the endoplasmicreticulum so that the final target protein could be accumulated in theendoplasmic reticulum. In addition, the HA epitope was used to confirmthe presence or absence of fusion protein expression by westernblotting. A diagram of the recombinant vector constructed in the presentexample is shown in FIG. 1 (M: M domain).

A nucleic acid sequence (SEQ ID NO: 10) of the recombinant vector shownin FIG. 1 is summarized in Table 2 below.

TABLE 2  Nucleic acid sequence (SEQ ID NO: 10) ofrecombinant vector for expressing a Leptin-M domain fusion domainNucleic acid sequence (5′→3′) XbaI tctaga (Restriction enzyme site) M17ggcgtgtgtgtgtgttaaaga (SEQ ID NO: 3) BiPAtggctcgctcgtttggagctaacagtaccgttgtgttggcgatcatcttcttcggtgagtgattttccgatcttcttctccgatttagatctcctctacattgttgcttaatctcagaaccttttttcgttgttcctggatctgaatgtgtttgtttgcaatttcacgatcttaaaaggttagatctcgattggtattgacgattggaatctttacgatttcaggatgtttatttgcgttgtcctctgcaatagaagaggctacgaagttaa (SEQ ID NO: 4) BamHI ggatccaagatgatgatgataag(Restriction (SEQ ID NO: 56) enzyme site) open reading frame Enk(Enterokinase cleavage site) Leptin gtgcctatccagaaagtccaggatggcaccaaagccctcatcaagaccattgtcaccaggatcaatgacatttcacacacgcagtcggtatccgccaagcagagggtcactggcttggacttcattcctgggcttcaccccattctgagtagtccaagatggaccagactctggcagtctatcaacaggtcctcaccagcctgccttcccaaaatgtgctgcagatagccaatgacctggagaatctccgagacctcctccatctgctggccttctccaagagctgctccctgcctcagaccagtggcctgcagaagccagagagcctggatggcgtcctggaagcctcactctactccacagaggtggtggctttgagcaggctgcagggctctctgcaggacattcttcaacagttggatgttagccctg aatgc (SEQ ID NO: 53) furincgtgtcaggcgaactagt cleavage site (SEQ ID NO: 57) SpeI (Restrictionenzyme site) M gcaaacatcactgtggattacttatatatagcaaactctaaatgttaatgagaatgtggaatgtggaaacaatacttgcacaaacaatgaggtgcataaccttacagaatgtaaaaatgcgtctgtaccatatctcataattca tgtactgctcctgat (SEQ ID NO: 1) HAtacccatacgatgttccagattacgct (SEQ ID NO: 58) linker tcc HDELcacgatgagctc (SEQ ID NO: 9) stop codon- tagctcgag XhoI

The N-glycosylation residues of an M domain of SEQ ID NO: 2 are asfollows:

1                            30 ANITVDYLYNKETKLFTAKLNVNENVECGN N1                          N2 31                           60NTCTNNEVHNLTECKNASVSISHNSCTAPD          N3    N4

Example 2. Confirmation of Increased Protein Expression byN-Glycosylation of M Domain

For comparison with an expression level of the fusion protein eLepfMproduced by the recombinant vector comprising the M domain, constructedin Example 1, a recombinant vector was constructed as follows.

A vector EeLepf in which the M domain was removed from the recombinantvector (EeLepfM; see FIG. 1) of Example 1 and a vector EeLepfM1234 inwhich four N-glycosylation sites (Asn) of the M domain (see Example 1)were mutated (mutation; Asn was substituted with Gln) were constructed(see Reference Example 1). The three vectors (see FIG. 2A) weretransformed into plant cells (see Reference Example 1) isolated fromleaves of Arabidopsis thaliana via polyethylene glycol (PEG), and thentreated with tunicamycin, which inhibits N-glycosylation, to confirm theeffect of N-glycosylation. After 24 hours, protein expression levelswere confirmed by western blotting. The results of the proteinexpression levels are shown in FIG. 2B (upper side: western blottingresults; lower side: a graph showing results of quantifying the proteinexpression levels obtained as a result of western blotting, by using anLAS4000 image analyzer (Error bars, standard deviation (n=3); *, p<0.05(Student's t-test)).

As illustrated in FIG. 2B, even when N-glycosylation did not occur, aprotein expression level was increased when the M domain was fused(EeLepfM+tunicamycin), but when tunicamycin was not added to therecombinant vector comprising the M domain (EeLepfM−tunicamycin),N-glycosylation occurred and the expression level of the target proteinwas increased to a maximum degree.

To more clearly verify whether the increase in protein expression leveldue to the fusion of the M domain was induced by N-glycosylation, arecombinant vector was constructed such that the M domain-free targetprotein (eLepf) and the M domain-fused protein (eLepfM) were targeted toeach of the endoplasmic reticulum, chloroplast, and mitochondria. Totarget the fusion proteins to each of the endoplasmic reticulum,chloroplast, and mitochondria, BiP (SEQ ID NO: 4), a Cab transit peptide(SEQ ID NO: 12), or F1-ATPase gamma subunit presequence (SEQ ID NO: 13)was fused to the N-terminal of the target protein, and an Mdomain-encoding nucleic acid sequence (SEQ ID NO: 1) was fused to theC-terminal of the target protein, thereby completing the construction ofthe recombinant vectors. Each recombinant vector was introduced into aplant cell using the above-described method, followed by culturing toexpress the corresponding fusion protein. Protein expression levels wereconfirmed by western blotting.

The results of the obtained protein expression levels are illustrated inFIG. 3 (upper side: a diagram of expression vectors; middle side:western blotting results; lower side: a graph showing results ofquantifying the protein expression levels obtained as a result ofwestern blotting by using an LAS4000 image analyzer (Error bars,standard deviation (n=3); *, p<0.05 (Student's t-test)).

As illustrated in FIG. 3, it was confirmed that, while the M domain-freeeLepf showed no difference in protein amount according to anintracellular organelle, in the case of the M domain-fused eLepfM, theexpression level of only EeLepfM targeted to the ER whereN-glycosylation occurs was increased.

When the results of FIGS. 2B and 3 are taken together, it was confirmedthat the increase in expression of the target protein due to the fusionof the M domain was caused by N-glycosylation.

Example 3. Expression Rate of M Domain-Fused Protein

As confirmed in Example 2, to understand the mechanism for theN-glycosylation-induced increase in protein expression, an expressionrate of the M domain-fused protein was examined. Each of the Mdomain-fused recombinant vector EeLepfM and the vector EeLepfM1234 inwhich the N-glycosylation sites of the M domain were mutated wastransformed into a plant cell, and after 12 hours, each vector wastreated with cycloheximide or dimethyl sulfoxide (DMSO), which blocksprotein synthesis, and then proteins were extracted at an interval of 12hours to perform western blotting thereon.

The results thereof are illustrated in FIG. 4 (upper side: westernblotting results; lower side: a graph showing results of quantifying theprotein expression levels obtained as a result of western blotting byusing an LAS4000 image analyzer (x-axis, time: Error bars, SD (n=3); *,p<0.05; ***, p<0.001).

As a result, as illustrated in FIG. 4, it was confirmed that there werealmost no significant difference in expression levels of EeLepfM andEeLepfM1234 at the initial time (12 h), but the difference continued toincrease over time, from which it was confirmed that the expression rateof EeLepfM was much faster than that of EeLepfM1234. However, it wasconfirmed that, while EeLepfM slightly disappeared in the plant cellwhen protein synthesis was blocked by treatment with cycloheximide,EeLepfM1234 was maintained as is up to 48 hours, from which it wasconfirmed that the fused protein EeLepfM1234 exhibited higher stabilityin the endoplasmic reticulum. These results indicate that a translationrate of a protein where N-glycosylation occurs is faster than that of aprotein where no N-glycosylation occurs.

Example 4. Fusion of Various Proteins and M Domain

To confirm whether the expression increase effect due to the M domain isapplicable to proteins other than the target protein (eLepf) used in theexamples, the M domain-encoding gene (SEQ ID NO: 1) was fused to anothertarget protein, e.g., a gene encoding Exendin4 (SEQ ID NO: 51) or a geneencoding GLP-1 (SEQ ID NO: 52), and a G domain, which is a translationenhancer domain, was fused thereto, thereby completing the constructionof a recombinant vector (see FIG. 1). Each recombinant vector wastransformed into a plant cell (see Reference Example 1), and thenprotein expression levels were confirmed by western blotting. Theresults thereof are illustrated in FIG. 5. As illustrated in FIG. 5, itwas confirmed that the protein expression level was significantlyincreased when N-glycosylation occurred, due to no treatment withtunicamycin, as compared to when treated with tunicamycin that blocksN-glycosylation.

Example 5. Fusion of Various Proteins and M Domain in Various Orders

In addition, a recombinant vector comprising leptin (Lep), aprotinin(Apr; SEQ ID NO: 11), or GFP (Gfp; SEQ ID NO: 15) at the position Xxx ofeach of the recombinant vectors illustrated in FIG. 6A was prepared(“(G4S)2”: linker (GGGGSGGGGS) (SEQ ID NO: 55)), and each recombinantvector was transformed into a plant cell, and then protein expressionlevels were confirmed by western blotting.

The results thereof are illustrated in FIG. 6B. As illustrated in FIG.6B, it was confirmed that, when a gene encoding a fusion protein of atarget protein and an M domain was expressed, an expression level of thetarget protein was significantly increased compared to when the M domainwas not fused, regardless of the type of target protein, an order linkedto the M domain, and the presence or absence of a linker.

Example 6: Test of Target Protein Expression According to Combinationsof N-Glycosylation Sites of M Domain

The expression level of a target protein (Leptin) was measured usingrecombinant vectors comprising genes encoding mutants (one mutation, twomutations, three mutations, and all four mutations of the fourN-glycosylation sites) into which Asn-Gln substitution mutation(s)was/were introduced to various combinations of the four N-glycosylationsites of the M domain (see the drawing of Example 1) (see FIGS. 7A-7C).Each recombinant vector was transformed into a protoplast of a plantcell, and then proteins were extracted, and the obtained proteinextracts were analyzed by western blotting using an anti-HA antibody.The signal intensity of bands obtained as a result of western blottingwas quantified using software provided with an LAS4000 image analyzer,and the results thereof are illustrated in FIGS. 7A-7C (on the x-axis,numbers following EeLepfM denote Asn-to-Gln mutated N-glycosylationsites). In FIGS. 7A-7C, the expression level of the target protein in acase in which each mutant was used was expressed as a relative valuewith respect to the expression level (1) of EeLepfM, which is anER-targeted wild type protein (fusion of wild-type M domain and targetprotein). FIG. 7A illustrates relative expression levels of the targetprotein when single Asn-Gln mutants were used, FIG. 7B illustratesrelative expression levels of the target protein when double Asn-Glnmutants were used, and FIG. 7C illustrates relative expression levels ofthe target protein when triple Asn-Gln mutants were used (Error bars, SD(n=4)).

As illustrated in FIGS. 7A-7C, when mutation occurred at positions 1 to3 of the N-glycosylation sites of the M domain, the expression level ofthe target protein was lower than that of wild type, but these casesexhibited a high expression level of the target protein compared to acase in which all four N-glycosylation sites were mutated. Whenconsidering that, even though a mutant in which all four N-glycosylationsites of the M domain were mutated was used, the expression level of thetarget protein was increased compared to a case in which the targetprotein was expressed alone (see FIG. 2B), even a case in which not onlya wild-type M domain but also a mutant M domain in which mutationsoccurred at positions 1 to 4 of the four N-glycosylation sites may beconsidered to contribute to increasing the expression level of thefusion-expressed target protein.

Example 7: Test for Correlation Between Low Expression Level ofUnglycosylated Protein and ER-Related Degradation

It was tested whether the low expression level of an unglycosylatedprotein is related to ER-associated degradation. In conclusion, it wasconfirmed that the low expression level of the unglycosylated proteinwas not due to ER-associated degradation.

More specifically, each of the EeLepfM vector (expression of a fusionprotein of Leptin and wild-type M domain) and EeLepfM1234 vector(expression of a fusion protein of Leptin and a mutant M domain in whichall four glycosylation sites were mutated (substituted with Gln) wastransformed into a plant protoplast, and then 20 μM MG132 (IUPAC name:BenzylN-[(2S)-4-methyl-1-[[(2S)-4-methyl-1-[[(2S)-4-methyl-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]carbamate),which is an inhibitor of 26S proteasome-mediated degradation, was addedto a protoplast culture medium at 18 h or 21 h, followed by furtherculturing of each protoplast for 6 hours or 3 hours.

[Schematic View of Test Design]

(HAT: Hours after Transformation)

For comparison (positive control), each protoplast was transformed withRbcs[T4, 7A]: GFP, and then treated with MG132 at 18 h and furthercultured for 6 hours. Rbcs[T4, 7A]: GFP, which is a GFP fusionconstruct, expresses a mutant form of the RbcS delivery peptidedefective in the introduction of a protein into the chloroplast, and isubiquitinated and degraded by the cytoplasmic 26S proteasome.

At 24 hours after transformation, proteins were extracted from eachtransformed protoplast, and then the protein extracts were analyzed bywestern blotting using an anti-HA antibody or an anti-GFP antibody.

The obtained western blotting results are illustrated in FIG. 8. Asillustrated in FIG. 8, the expression level of Rbcs[T4, 7A]: GFP washigher upon treatment with MG132, compared to non-treatment with MG132,and these results show that MG132 inhibits 26S proteasome-dependentprotein degradation under test conditions of the present example.However, the expression levels of the EeLepfM and EeLepfM1234 proteinsshowed no difference regardless of MG132 treatment, which indicates thatER-associated degradation did not play a role in the expression of theN-glycosylated EeLepfM protein and the unglycosylated EeLepfM1234protein.

Example 8: Test for Effect of N-Glycosylation of Target Protein Itselfand N-Glycosylation of M Domain on Target Protein Expression

It was examined whether the expression level of the target protein isincreased even in the case of N-glycosylation of the target proteinitself that was not fused with the M domain, that is, a case in whichN-glycosylation of the target protein itself was possible due to itsintrinsic inclusion of N-glycosylation sites.

To this end, with reference to the previous examples, an LIF(EeLiff)-expressing recombinant vector to which the M domain was fusedor not fused was constructed using a leukemia inhibitory factor (LIF)(SEQ ID NO: 17; nucleic acid-encoding sequence: SEQ ID NO: 16; EeLiff;intrinsically having 6 N-glycosylation sites) instead of the Leptinprotein, each recombinant vector was transformed into a plantprotoplast, and after 24 hours, proteins were extracted and proteinexpression levels were measured by western blotting.

The obtained protein expression levels are illustrated in FIG. 9. Asillustrated in FIG. 9, it can be seen that the expression level of Mdomain-fused EeLiffM is significantly high compared to that of EeLiff towhich the M domain was not fused (RbcL: loading control). These resultsshow that, while N-glycosylation at N-glycosylation sites intrinsicallycomprised in the target protein does not affect the expression level ofthe target protein, N-glycosylation of the M domain, which is a fusionpartner fused with the target protein, plays an important role in theexpression level of the target protein.

Example 9: Expression Level of Fusion Protein Fused with Portion orExtension Portion of M Domain

The expression level of a fusion protein in which the target protein(Leptin) was fused with a portion or extension portion of the M domainwas tested. To this end, with reference to the method of Example 2, afusion protein in which the target protein and a fragment (SEQ ID NO: 6)having a length of 20 amino acids at positions 41-60 of the M domain(SEQ ID NO: 2; total 60aa) (EeLepfM20; comprising 1 N-glycosylation site(N4: Asn at position 46), a fusion protein in which a fragment (SEQ IDNO: 7) having a length of 40 amino acids at positions 21-60 was fused(EeLepfM40; comprising 3 N-glycosylation sites of the M domain (N2: Asnat position 30; N3: Asn at position 40; and N4: Asn at position 46), anda fusion protein (E3LepfM80) in which a fragment (SEQ ID NO: 8) having alength of a total of 80 amino acids which extends by 10 amino acidstowards each of the N-terminal and C-terminal of the M domain of SEQ IDNO: 2 in CD45 (SEQ ID NO: 5) were each introduced into a protoplast of aplant cell and expressed, and then proteins were extracted andexpression levels thereof were measured by western blotting. Forcomparison, the expression level of EeLepf to which the M domain was notfused was also measured.

The obtained protein expression levels are illustrated in FIG. 10. Asillustrated in FIG. 10, all of EeLepfM20, EeLepfM40, EeLepfM60, andEeLepfM80 exhibited higher expression levels than that of EeLepf(EeLepf<<EeLepfM20<EeLepfM40≈EeLepfM≈EeLepfM80).

Example 10: Test for Transcript Level According to Fusion of M Domain orVarious M Domain Mutants

A plant cell (Reference Example 1) was transformed with each ofrecombinant vectors comprising a wild-type (not mutated) M domain orvarious M domain mutants in which each of the N-glycosylation sites wasmutated and leptin, and total RNA was extracted after 1 day to performquantitative RT-PCR. For a detailed method, refer to Reference Example4.

The obtained RNA levels are illustrated in FIG. 11. FIG. 11 illustratesmean values of mRNA levels in the case in which each M domain mutant wasfused, relative to an mRNA level (=1) in the case in which a wild-type(not mutated) M domain (i.e., fully glycosylated) and leptin were fusedwith each other (Error bar, SD (n=3 for M to 14; n=2 for 23 to 1234). Asa result of a Student's t-test, p values were equal to or greater than0.05, which indicates that there was no difference in mRNA level betweenM domain-fused leptin and leptins to which mutated M domains were fused.These results indicate that the increase in expression due to fusion ofthe M domain is not due to an increase in mRNA level by increasingtranscription, and suggests that such an expression increase is due tothe promotion of translation from mRNA into a protein.

The invention claimed is:
 1. A method of producing a target protein, themethod comprising culturing a recombinant cell comprising a recombinantvector for expressing the target protein, the recombinant vectorcomprising a gene encoding a fusion protein comprising the targetprotein and an N-glycosylation domain fused to the C-terminal orN-terminal of the target protein, wherein the N-glycosylation domain isa polypeptide having a length of 10 to 60 consecutive amino acids in ahuman CD45 protein of SEQ ID NO: 5, the polypeptide consisting of ahuman CD45-derived M domain of SEQ ID NO: 2 or a portion of the Mdomain, wherein the portion of the M domain is a polypeptide fragment of10 or more amino acids and one or more N-glycosylation sites selectedfrom Asn at position 2, Asn at position 30, Asn at position 40, and Asnat position 46, of SEQ ID NO:
 2. 2. The method of claim 1, wherein thepolypeptide fragment selected from the group consisting of “LTECKNASVSISHNSCTAPD (SEC) ID NO: 6)” and “NVNENVECGN NTCTNNEVHN LTECKNASVSISHNSCTAPD (SEQ ID NO: 7).
 3. The method of 1, wherein the polypeptidefragment is “NVNENVECGN NTCTNNEVHN LTECKNASVS ISHNSCTAPD (SEQ ID NO:7)”.
 4. The method of 1, wherein the fusion protein further comprises apeptide linker between the target protein and the N-glycosylationdomain.
 5. The method of 1, wherein the fusion protein is targeted to anendoplasmic reticulum.
 6. The method of 1, wherein the recombinantvector further comprises a BiP (chaperone binding protein)-encodinggene, a HDEL (His-Asp-Glu-Leu) (SEQ ID NO: 54) peptide-encoding gene, ora combination thereof.
 7. The method of 1, wherein the recombinantvector is for use in expression in a plant cell.
 8. A method ofproducing a target protein, the method comprising growing a transgenicorganism comprising a recombinant cell comprising a recombinant vectorfor expressing the target protein, the recombinant vector comprising agene encoding a fusion protein comprising the target protein and anN-glycosylation domain fused to the C-terminal or N-terminal of thetarget protein, wherein the N-glycosylation domain is a polypeptidehaving a length of 10 to 60 consecutive amino acids in a human CD45protein of SEQ ID NO: 5, the polypeptide consisting of a humanCD45-derived M domain of SEQ ID NO: 2 or a portion of the M domain,wherein the portion of the M domain is a polypeptide fragment of 10 ormore amino acids and one or more N-glycosylation sites selected from Asnat position 2, Asn at position 30, Asn at position 40, and Asn atposition 46, of SEQ ID NO:
 2. 9. The method of claim 8, the polypeptidefragment selected from the group consisting of “LTECKNASVS ISHNSCTAPD(SEQ ID NO: 6)” and “NVNENVECGN NTCTNNEVHN LTECKNASVS ISHNSCTAPD (SEQ IDNO: 7).
 10. The method of 8, wherein the polypeptide fragment is“NVNENVECGN NTCTNNEVHN LTECKNASVS ISHNSCTAPD (SEQ ID NO: 7)”.
 11. Themethod of 8, wherein the fusion protein further comprises a peptidelinker between the target protein and the N-glycosylation domain. 12.The method of 8, wherein the fusion protein is targeted to anendoplasmic reticulum.
 13. The method of 8, wherein the recombinantvector further comprises a BiP (chaperone binding protein)-encodinggene, a HDEL (His-Asp-Glu-Leu) (SEQ ID NO: 54) peptide-encoding gene, ora combination thereof.
 14. The method of 8, wherein the recombinantvector is for use in expression in a plant cell.